Immunogenicity and safety of inactivated SARS-CoV-2 vaccine in haemodialysis patients: a prospective cohort study

End-stage renal disease patients on haemodialysis (HD) have been largely excluded from SARS-CoV-2 vaccine trials due to safety reasons and shown to mount lower responses to vaccination. This study aims to evaluate the immunogenicity and safety of inactivated COVID-19 vaccine among HD patients compared to healthy controls. All subjects who received the primary inactivated COVID-19 vaccination had their blood samples tested 21 days after the second dose. We report the immunogenicity based on anti-RBD IgG titre (IU/mL), the inhibition rate of neutralizing antibodies (NAbs) (%) to RBD, and seroconversion rates. Adverse events were assessed within 30 min and on the 7th day after each dose. Among 75 HD patients and 71 healthy controls, we observed no significant difference in all immunogenicity measures: anti-RBD IgG GMT (277.91 ± 7.13 IU/mL vs. 315.50 ± 3.50 IU/mL, p = 0.645), NAbs inhibition rate (82% [53–96] vs. 84% [39–98], p = 0.654), and seroconversion rates (anti-RBD IgG: 86.7% vs. 85.9%, p = 0.895; NAbs: 45.3% vs. 60.6%, p = 0.065). The number of adverse events is not significantly different between the two groups. The primary inactivated SARS-CoV-2 vaccination elicits an adequate antibody response and can be safely administered in haemodialysis patients.


Methods
Study design. An observational prospective cohort study was conducted among maintenance haemodialysis patients and healthy individuals who received the primary inactivated COVID-19 vaccination (CoronaVac®) during the national mass vaccination program in the Special Region of Yogyakarta and Central Java between July 2021 to October 2021. The timeline of this study is presented in Fig. 1. We recruited haemodialysis patients from four outpatient dialysis centres in Yogyakarta and Central Java, Indonesia. Subjects were vaccinated with two doses of inactivated SARS-CoV-2 vaccine (CoronaVac, 3 µg of inactivated whole-virus SARS-CoV-2 in 0.5 ml) 28 days apart. We evaluated the anti-RBD IgG antibody titre, inhibition rate (%inhibition) of NAbs to RBD of SARS-CoV-2, and seroconversion rates in HD patients compared to healthy controls. We also monitored and compared local and systemic adverse events in both groups. Seroconversion is defined as the conversion from seronegative (NAbs %inhibition < 30%) at baseline to seropositive (NAbs %inhibition ≥ 30%). Blood samples were collected twice: before vaccination and 21 days after the second dose. All participants were monitored for solicited and unsolicited AEs within 30 and on the 7th day after each dose by phone call.
Patient selection. Generally, we included subjects aged between 18 and 59 years old and excluded those with symptoms of respiratory tract infections within the past week, evidence of ongoing infections, and history of COVID-19. HD patients were included if they had undergone routine haemodialysis twice weekly for at least three months with an arteriovenous (AV) fistula or graft as the vascular access. All HD patients in our dialysis centres undergo haemodialysis only twice weekly due to limited resources. Patients with double-lumen catheters (DLC) as the vascular access were excluded to acquire a homogenous sample. Moreover, AV fistula has been shown to contribute to better survival among HD patients with COVID-19 11 . Healthy subjects with a history of kidney diseases was excluded. Any subjects were excluded if they have acute conditions related to ESRD

Immunogenicity. The immunogenicity among participants after inactivated SARS-CoV-2 vaccination is
measured with anti-RBD geometric mean titre (GMT), NAbs percentage of inhibition, and seroconversion rates at 21 days after the second dose (Table 2 and Fig. 3). There was no significant difference between both groups in anti-RBD IgG GMT, NAbs percentage of inhibition, and seroconversion rates after the second dose. However, the increase in the inhibition rate of healthy controls (46%) was significantly more significant than in HD patients (25%) (p = 0.049). The change in log anti-RBD IgG and NAbs %inhibition before and after vaccination among both groups is illustrated in Fig. 4. Considering that many participants were already seropositive at baseline (Table 1), the immunogenicity measures might be confounded. Therefore, we conducted further analyses to compare HD patients and seronegative controls at baseline ( Table 3). The seroconversion rate of NAbs is the only significantly different measure between the HD patients (34/42 or 81%) and controls (43/43 or 100%) (p = 0.003).

Responders vs. non-responders.
Overall, the seroconversion rate among all participants is 86.3% (126/146) and 52.74% (77/146) for NAbs. Vaccination responder rates were respectively 86.7% (65/75) in HD patients and 85.9% (61/71) in healthy controls for anti-RBD IgG antibody, and 45.3% (34/75) in HD patients and 60.6% (43/71) in healthy controls for NAbs. Table 4, compares the clinical and laboratory characteristics between responders and non-responders. Hypertension is significantly associated with a lower seroconversion rate of neutralizing antibodies in the bivariate analysis (p = 0.021). Further multivariate analysis with binary logistic  www.nature.com/scientificreports/ regression is conducted for variables with a p-value of < 0.25, i.e., hypertension, haemoglobin, and platelet count (Table 5). Hypertension, haemoglobin, and platelet count are not significantly different between NAbs responders and non-responders. Meanwhile, this further analysis could not be done for anti-RGB IgD antibody because no variables generated a p-value of < 0.25 in the bivariate analysis.
Adverse events. The occurrence of adverse events after the first and second doses of vaccination among HD patients and healthy controls are summarized in Table 6 and Fig. 5. Overall, there is no difference in the number of adverse events between HD patients and healthy controls, except for solicited AEs after the second dose, which was higher among HD patients. The most common AEs included pain at the injection site (21-33%), fatigue (6-16%), headache (5-13%), muscle aches (1-9%), and fever (0-8%) (Fig. 5). The AEs were almost entirely resolved on the 7th day. Almost all AEs were classified as Grade 1 or Grade 2. No serious adverse events were reported among both groups.

Discussion
This study evaluated the immunogenicity and safety after vaccination with inactivated COVID-19 vaccines among haemodialysis patients in comparison with healthy individuals. Antibody levels have been demonstrated to peak around 2-4 weeks after vaccination. We assessed the humoral response three weeks after the second dose since HD patients may have delayed immune response. As expected, in terms of baseline characteristics, several comorbid conditions, including diabetes mellitus, hypertension, and anaemia, are significantly more prevalent in HD patients compared to healthy controls. Diabetes mellitus and hypertension are the leading causes of chronic kidney disease (CKD) 16 . Meanwhile, anaemia is a common complication in CKD, and the prevalence increases with the stage of CKD, from 8.4% at stage 1 to 53.4% at stage 5 17 . All laboratory values are significantly different among both groups owing to the pathophysiological processes and complications involved in compromised kidney function. Our study demonstrated no significant difference in anti-RBD IgG geometric mean titre, NAbs median inhibition rate, and seroconversion rates after two doses of vaccination with inactivated COVID-19 vaccines (CoronaVac®) between HD patients and healthy individuals. However, several study participants (44% of HD patients and 39.4% of healthy controls) were seropositive before vaccination, which might confound the results. These seropositive subjects might have had previous asymptomatic to mild COVID-19 and were not tested and diagnosed. Being an archipelagic state and the world's fourth-most populous country, COVID-19 tracing, and testing in Indonesia were not adequate and thorough, especially during the early pandemic. Therefore, we also evaluated the increase in NAbs inhibition rate and found that this value was significantly lower among HD patients than healthy controls. We also conducted further analyses to compare only HD patients and among healthy controls who were seronegative at baseline. In this subgroup, the seroconversion rate of NAbs was significantly lower among HD patients than among healthy controls. These findings demonstrated an adequate but weaker humoral response after vaccination in HD patients than in healthy controls.
Several previous studies have demonstrated similar results. Murt et al. evaluated the immunologic response toward inactivated vaccine (CoronaVac®) and mRNA vaccine (BNT162b2) in haemodialysis patients compared to healthy healthcare workers. The study also demonstrated significantly lower seropositivity rates and antibody  Our study also demonstrated that no specific clinical or laboratory characteristics are associated with vaccine responsiveness. This is inconsistent with several previous studies' findings, which reported lower antibody response and vaccine effectiveness in individuals with hypertension 19,[22][23][24] . Recent evidence suggests that certain types of hypertension may be associated with the adaptive immune system. For instance, a significant change in T cell immunometabolism can modulate the metabolic processes and eventually lead to aberrant T cell activation, differentiation, and proliferation, which contribute to the pathogenesis of hypertension [25][26][27] . In previous studies, the immunogenicity of COVID-19 vaccines has been demonstrated to be lower among patients with diabetes mellitus 19,28 .
Overall, the occurrence of adverse events after vaccination is not significantly different between HD patients and healthy controls, except for the second dose solicited AEs rate being higher among HD patients. This finding contradicts several previous studies, demonstrating that adverse events rates are higher in healthy individuals 29 . Simon et al. revealed a significantly higher number of local and systemic adverse events in healthy controls than in HD patients after both vaccine doses. Similar to our finding, no serious adverse events were reported in either group 30 . Park et al. demonstrated that the proportion of participants with injection site swelling, heat sensation at the injection site, headache, and fatigue were higher within seven days of the first dose among healthy people than among HD patients. Meanwhile, there was no significant difference in adverse events after the second dose between HD patients and healthy people 31 . Similarly, Kolb et al. also reported no substantial differences in adverse events between HD patients and healthy controls after vaccination with BNT162b2 or mRNA-1273 32 . www.nature.com/scientificreports/ The strengths of our study are the prospective cohort design and the use of inactivated COVID-19 vaccine when many other studies utilize mRNA or vector-based vaccines. This study is possibly one of the few studies examining the immune response in haemodialysis patients after inactivated SARS-CoV-2 vaccination. Our study had several limitations, including a relatively small number of participants and the inability to appropriately exclude all participants with past SARS-CoV-2 infections due to inadequate tracing and testing in Indonesia during the early pandemic. Past SARS-CoV-2 infections might confound the level of immunity mounted by the vaccine recipients. The study was conducted at only four centres in the Special Region of Yogyakarta and Central Java. Therefore, the results of this study cannot be generalized to all HD populations. Moreover, the sVNT is a surrogate test for neutralizing antibodies, which is less standardized than the plaque reduction neutralization test. Hence, we cannot unequivocally conclude the assumption of protective immunity against infection after this vaccine regimen.
The results of our study suggest that further studies on the effect of a booster dose should be conducted to promote a more substantial and persistent antibody response in HD patients. In addition, patients with low or no response might benefit from regular antibody measurement and more intensive vaccination schedules.

Conclusions
After two doses of inactivated SARS-CoV-2 vaccination, we report an adequate antibody response among haemodialysis patients. Inactivated SARS-CoV-2 vaccine can be safely administered to haemodialysis patients with tolerable adverse events.

Data availability
The datasets generated and analysed during the current study are not publicly available because of privacy and ethical restrictions. Still, anonymized data are available from the corresponding author at reasonable request.